Multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry

ABSTRACT

This invention provides methods for detecting single nucleotide polymorphisms and multiplex genotyping using dideoxynucleotides and mass spectrometry.

This application is a continuation-in-part and claims priority of U.S.Ser. No. 10/194,882, filed Jul. 12, 2002, the contents of which arehereby incorporated by reference into this application.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced inparentheses. Citations for these references may be found at the end ofthe specification immediately preceding the claims. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application to more fully describe the state of theart to which this invention pertains.

Single nucleotide polymorphisms (SNPs), the most common geneticvariations in the human genome, are important markers for identifyingdisease genes and for pharmacogenetic studies (1, 2). SNPs appear in thehuman genome with an average density of once every 1000-base pairs (3).To perform large-scale SNP genotyping, a rapid, precise andcost-effective method is required. Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)(4) allows rapid and accurate sample measurements (5-7) and has beenused in a variety of SNP detection methods including hybridization(8-10), invasive cleavage (11, 12) and single base extension (SBE) (5,13-17). SBE is widely used for multiplex SNP analysis. In this method,primers designed to anneal immediately adjacent to a polymorphic siteare extended by a single dideoxynucleotide that is complementary to thenucleotide at the variable site. By measuring the mass of the resultingextension product, a particular SNP can be identified. Current SBEmethods to perform multiplex SNP analysis using MS require unambiguoussimultaneous detection of a library of primers and their extensionproducts. However, limitations in resolution and sensitivity ofMALDI-TOF MS for longer DNA molecules make it difficult tosimultaneously measure DNA fragments over a large mass range (6). Therequirement to measure both primers and their extension products in thisrange limits the scope of multiplexing.

A high fidelity DNA sequencing method has been developed which usessolid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs)by detection with fluorescence (18) or mass spectrometry (19),eliminating false terminations and excess primers. Combinatorialfluorescence energy transfer tags and biotin-ddNTPs have also been usedto detect SNPs (20).

False stops or terminations occur when a deoxynucleotide rather than adideoxynucleotide terminates a se+quencing fragment. It has been shownthat false stops and primers which have dimerized can produce peaks inthe mass spectra that can mask the actual results preventing accuratebase identification (21).

The present application discloses an approach using solid phasecapturable biotin-ddNTPs in SBE for multiplex genotyping by MALDI-TOFMS. In this method primers that have different molecular weights andthat are specific to the polymorphic sites in the DNA template areextended with biotin-ddNTPs by DNA polymerase to generate3′-biotinylated DNA extension products. The 3′-biotinylated DNAs arethen captured by streptavidin-coated magnetic beads, while theunextended primers and other components in the reaction are washed away.The pure DNA extension products are subsequently released from themagnetic beads, for example by denaturing the biotin-streptavidininteraction with formamide, and analyzed with MALDI-TOF MS. Thenucleotide at the polymorphic site is identified by analyzing the massdifference between the primer extension product and an internal massstandard added to the purified DNA products. Since the primer extensionproducts are isolated prior to MS analysis, the resulting mass spectrumis free of non-extended primer peaks and their associated dimers, whichincreases the accuracy and scope of multiplexing in SNP analysis. Thesolid phase purification system also facilitates desalting of thecaptured oligonucleotides. Desalting is critical in sample preparationfor MALDI-TOF MS measurement since alkaline and alkaline earth salts canform adducts with DNA fragments that interfere with accurate peakdetection (21).

SUMMARY OF THE INVENTION

This invention is directed to a method for determining the identity of anucleotide present at a predetermined site in a DNA whose sequenceimmediately 3′ of such predetermined site is known which comprises:

-   -   (a) treating the DNA with an oligonucleotide primer whose        sequence is complementary to such known sequence so that the        oligonucleotide primer hybridizes to the DNA and forms a complex        in which the 3′ end of the oligonucleotide primer is located        immediately adjacent to the predetermined site in the DNA;    -   (b) simultaneously contacting the complex from step (a) with        four different labeled dideoxynucleotides, in the presence of a        polymerase under conditions permitting a labeled        dideoxynucleotide to be added to the 3′ end of the primer so as        to generate a labeled single base extended primer, wherein each        of the four different labeled dideoxynucleotides (i) is        complementary to one of the four nucleotides present in the DNA        and (ii) has a molecular weight which can be distinguished from        the molecular weight of the other three labeled        dideoxynucleotides using mass spectrometry; and    -   (c) determining the difference in molecular weight between the        labeled single base extended primer and the oligonucleotide        primer so as to identify the dideoxynucleotide incorporated into        the single base extended primer and thereby determine the        identity of the nucleotide present at the predetermined site in        the DNA.

In one embodiment, the method further comprises after step (b) the stepsof:

-   -   (i) contacting the labeled single base extended primer with a        surface coated with a compound that specifically interacts with        a chemical moiety attached to the dideoxynucleotide by a linker        so as to thereby capture the extended primer on the surface; and    -   (ii) treating the labeled single base extended primer so as to        release it from the surface.

In one embodiment, the method further comprises after step (i) the stepof treating the surface to remove primers that have not been extended bya labeled dideoxynucleotide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Scheme of single base extension for multiplex SNP analysis usingbiotin-ddNTPs and MALDI-TOF MS. Primers that anneal immediately next tothe polymorphic sites in the DNA template are extended by DNA polymeraseof a biotin-ddNTP in a sequence-specific manner. After solid phasecapture and isolation of the 3′-biotinylated DNA extension fragments,MALDI-TOF MS was used to analyze these DNA products to yield a massspectrum. From the relative mass of each extended primer, compared tothe mass of an internal standard, the nucleotide at the polymorphic siteis identified.

FIG. 2. Multiplex SNP genotyping mass spectra generated usingbiotin-ddNTPs. Inset is a magnified view of heterozygote peaks. Massesof the extension product in reference to the internal mass standard werelisted on each single base extension peak. The mass values inparenthesis indicate the mass difference between the extension productsand the corresponding primers. (A) Detection of six nucleotidevariations from synthetic DNA templates mimicking mutations in the p53gene. Four homozygous (T, G, C and C) and one heterozygous (C/A)genotypes were detected. (B) Detection of two heterozygotes (A/G andC/G) in the human HFE gene.

FIG. 3: Structure of four mass tagged biotinylated ddNTPs. Any of thefour ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) can be used with any of theillustrated linkers.

FIG. 4: Synthesis scheme for mass tag linkers. For illustrativepurposes, the linkers are labeled to correspond to the specific ddNTPwith which they are shown coupled in FIGS. 3, 5, 7, 8 and 9. However,any of the three linkers can be used with any ddNTP. (i) (CF₃CO)₂O; (ii)Disuccinimidylcarbonate/diisopropylethylamine; (iii) Propargyl amine.

FIG. 5: The synthesis of ddATP-Linker-II-11-Biotin. (i) Linker II,tetrakis(triphenylphosphine) palladium(0); (ii) POCl₃, Bn₄N⁺pyrophosphate; (iii) NH₄OH; (iv) Sulfo-NHS-LC-Biotin.

FIG. 6: DNA products are purified by a streptavidin coated porous silicasurface. Only the biotinylated fragments are captured. These fragmentsare then cleaved by light irradiation (hv) to release the capturedfragments, leaving the biotin moiety still bound to the streptavidin.

FIG. 7: Mechanism for the cleavage of photocleavable linkers.

FIG. 8: The structures of ddNTPs linked to photocleavable (PC) biotin.Any of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) can be used with anyof the shown linkers.

FIG. 9: The synthesis of ddATP-Linker-II-PC-Biotin. PC=photocleavable.

FIG. 10: Schematic for capturing a DNA fragment terminated with adideoxynucleoside monophosphate on a surface. The dideoxynucleosidemonophosphate (ddNMP) which is on the 3′ end of the DNA fragment isattached via a linker to a chemical moiety “X” which interacts with acompound “Y” on the surface to capture the DNA fragment terminated withthe ddNMP. The DNA fragment can be freed from the surface either bydisrupting the interaction between chemical moiety X and compound Y(lower scheme) or by cleaving the linker (upper scheme).

FIGS. 11A-11C: Schematic of a high throughput channel based purificationsystem. Sample solutions can be pushed back and forth between the twoplates through glass capillaries and the streptavidin coated channels inthe chip. The whole chip can be irradiated to cleave the samples afterimmobilization.

FIG. 12: The synthesis of streptavidin coated porous surface.

FIGS. 13A-13C: Simultaneous detection of nucleotide variations in 30codons of the p53 tumor suppressor gene by MALDI-TOF MS using solidphase capturable biotinylated dideoxynucleotide. Each peak represents adifferent polymorphism labeled with its nucleotide identity and absolutemass value. The value in parentheses, denoting the mass differencebetween each DNA extension product and its corresponding primer, is usedto determine the nucleotide identity. (A) A mass spectrum from a Wilms'tumor sample showing 30 wild type p53 sequences. (B) A mass spectrumfrom a head and neck tumor (primary tumor biopsy) containing aheterozygous genotype G/T (4684/4734 Da) (boxed) in codon 157,corresponding to the wild type and mutant alleles, respectively. (C) Amass spectrum from a colorectal tumor cell line (HT-29) containing ahomozygous G to A mutation (boxed) in codon 273 of the p53 gene. Thecolorectal tumor cell line (SW-480) contained the identical G to Amutation in codon 273.

FIGS. 14A-14B: (A) A mass spectrum from a head and neck tumor sampleshowing 30 wild type sequences of the p53 gene. (B) A mass spectrum froma head and neck tumor cell line (SCC-4) containing a homozygous C (5881Da) to T (5970 Da) mutation (boxed) in codon 151 of the p53 gene. Bothspectra were produced using the primers shown in Table 3 with primer 16replaced by primer 5′-TGTGGGTTGATTCCACA-3′ for detecting the variationin codon 151 (C/TCC).

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are presented as an aid in understanding thisinvention.

The standard abbreviations for nucleotide bases are used as follows:adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).

A nucleotide analogue refers to a chemical compound that is structurallyand functionally similar to the nucleotide, i.e. the nucleotide analoguecan be recognized by polymerase as a substrate. That is, for example, anucleotide analogue comprising adenine or an analogue of adenine shouldform hydrogen bonds with thymine, a nucleotide analogue comprising C oran analogue of C should form hydrogen bonds with G, a nucleotideanalogue comprising G or an analogue of G should form hydrogen bondswith C, and a nucleotide analogue comprising T or an analogue of Tshould form hydrogen bonds with A, in a double helix format.

This invention is directed to a method for determining the identity of anucleotide present at a predetermined site in a DNA whose sequenceimmediately 3′ of such predetermined site is known which comprises:

-   -   (a) treating the DNA with an oligonucleotide primer whose        sequence is complementary to such known sequence so that the        oligonucleotide primer hybridizes to the DNA and forms a complex        in which the 3′ end of the oligonucleotide primer is located        immediately adjacent to the predetermined site in the DNA;    -   (b) simultaneously contacting the complex from step (a) with        four different labeled dideoxynucleotides, in the presence of a        polymerase under conditions permitting a labeled        dideoxynucleotide to be added to the 3′ end of the primer so as        to generate a labeled single base extended primer, wherein each        of the four different labeled dideoxynucleotides (i) is        complementary to one of the four nucleotides present in the DNA        and (ii) has a molecular weight which can be distinguished from        the molecular weight of the other three labeled        dideoxynucleotides using mass spectrometry; and    -   (c) determining the difference in molecular weight between the        labeled single base extended primer and the oligonucleotide        primer so as to identify the dideoxynucleotide incorporated into        the single base extended primer and thereby determine the        identity of the nucleotide present at the predetermined site in        the DNA.

In one embodiment, each of the four labeled dideoxynucleotides comprisesa chemical moiety attached to the dideoxynucleotide by a differentlinker which has a molecular weight different from that of each otherlinker.

In one embodiment, the method further comprises after step (b) the stepsof:

-   -   (i) contacting the labeled single base extended primer with a        surface coated with a compound that specifically interacts with        a chemical moiety attached to the dideoxynucleotide by a linker        so as to thereby capture the extended primer on the surface; and    -   (ii) treating the labeled single base extended primer so as to        release it from the surface.

In a further embodiment, the method comprises after step (i) the step oftreating the surface to remove primers that have not been extended by alabeled dideoxynucleotide and any non-captured component.

In one embodiment of the method step (c) comprises determining thedifference in mass between the labeled single base extended primer andan internal mass calibration standard added to the extended primer. Inone embodiment, the internal mass standard is 5′-TTTTTCTTTTTCT-3′ (SEQID NO: 5) (MW=3855 Da).

In one embodiment, the chemical moiety is attached via a differentlinker to different dideoxynucleotides. In one embodiment, the differentlinkers increase mass separation between different labeled single baseextended primers and thereby increase mass spectrometry resolution.

In one embodiment, the dideoxynucleotide is selected from the groupconsisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP),2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate(ddTTP).

In different embodiments of the methods described herein, theinteraction between the chemical moiety attached to thedideoxynucleotide by the linker and the compound on the surfacecomprises a biotin-streptavidin interaction, a phenylboronicacid-salicylhydroxamic acid interaction, or an antigen-antibodyinteraction.

In one embodiment, the step of releasing the labeled single baseextended primer from the surface comprises disrupting the interactionbetween the chemical moiety attached by the linker to thedideoxynucleotide and the compound on the surface. In differentembodiments, the interaction is disrupted by a means selected from thegroup consisting of one or more of a physical means, a is chemicalmeans, a physical chemical means, heat, and light. In one embodiment,the interaction is disrupted by light. In one embodiment, theinteraction is disrupted by ultraviolet light. In different embodiments,the interaction is disrupted by ammonium hydroxide, formamide, or achange in pH (-log H⁺ concentration).

In different embodiments, the linker can comprise a chain structure, ora structure comprising one or more rings, or a structure comprising achain and one or more rings. In different embodiments, thedideoxynucleotide comprises a cytosine or a thymine with a 5-position,or an adenine or a guanine with a 7-position, and the linker is attachedto the dideoxynucleotide at the 5-position of cytosine or thymine or atthe 7-position of adenine or guanine.

In different embodiments, the step of releasing the labeled single baseextended primer from the surface comprises cleaving the linker betweenthe chemical moiety and the dideoxynucleotide. In different embodiments,the linker is cleaved by a means selected from the group consisting ofone or more of a physical means, a chemical means, a physical chemicalmeans, heat, and light. In one embodiment, the linker is cleaved bylight. In one embodiment, the linker is cleaved by ultraviolet light. Indifferent embodiments, the linker is cleaved by ammonium hydroxide,formamide, or a change in pH (-log H⁺ concentration);

In one embodiment, the linker comprises a derivative of 4-aminomethylbenzoic acid. In one embodiment, the linker comprises a 2-nitrobenzylgroup or a derivative of a 2-nitrobenzyl group. In one embodiment, thelinker comprises one or more fluorine atoms.

In one embodiment, the linker is selected from the group consisting of:

In one embodiment, a plurality of different linkers is used to increasemass separation between different labeled single base extended primersand thereby increase mass spectrometry resolution.

In one embodiment, the chemical moiety comprises biotin, the labeleddideoxynucleotide is a biotinylated dideoxynucleotide, the labeledsingle base extended primer is a biotinylated single base extendedprimer, and the surface is a streptavidin-coated solid surface. In oneembodiment, the biotinylated dideoxynucleotide is selected from thegroup consisting of ddATP-11-biotin, ddCTP-11-biotin, ddGTP-11-biotin,and ddTTP-16-biotin.

In one embodiment, the biotinylated dideoxynucleotide is selected fromthe group consisting of:

wherein ddNTP1, ddNTP2, ddNTF3, and ddNTP4 represent four differentdideoxynucleotides, or their analogues.

In one embodiment, the biotinylated dideoxynucleotide is selected fromthe group consisting of:

In one embodiment, the biotinylated dideoxynucleotide is selected fromthe group consisting of:

wherein ddNTP1, ddNTP2, ddNTP3, and ddNTP4 represent four differentdideoxynucleotides or their analogues.

In one embodiment, the biotinylated dideoxynucleotide is selected fromthe group consisting of:

In one embodiment, the streptavidin-coated solid surface is astreptavidin-coated magnetic bead or a streptavidin-coated silica glass.

In one embodiment of the method, steps (a) and (b) are performed in asingle container or in a plurality of connected containers.

The invention provides methods for determining the identity ofnucleotides present at a plurality of predetermined sites, whichcomprises carrying out any of the methods disclosed herein using aplurality of different primers each having a molecular weight differentfrom that of each other primer, wherein a different primer hybridizesadjacent to a different predetermined site. In one embodiment, differentlinkers each having a molecular weight different from that of each otherlinker are attached to the different dideoxynucleotides to increase massseparation between different labeled single base extended primers andthereby increase mass spectrometry resolution.

In one embodiment, the mass spectrometry is matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

Linkers are provided for attaching a chemical moiety to adideoxynucleotide, wherein the linker comprises a derivative of4-aminomethyl benzoic acid.

In one embodiment, the dideoxynucleotide is selected from the groupconsisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP),2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate(ddTTP).

In one embodiment, the linker comprises one or more fluorine atoms.

In one embodiment, the linker is selected from the group consisting of:

In different embodiments, the linker can comprise a chain structure, ora structure comprising one or more rings, or a structure comprising achain and one or more rings.

In different embodiments, the linker is cleavable by a means selectedfrom the group consisting of one or more of a physical means, a chemicalmeans, a physical chemical means, heat, and light. In one embodiment,the linker is cleavable by ultraviolet light. In different embodiments,the linker is cleavable by ammonium hydroxide, formamide, or a change inpH (-log H⁺ concentration).

In different embodiments of the linker, the chemical moiety comprisesbiotin, streptavidin or related analogues that have affinity withbiotin, phenylboronic acid, salicylhydroxamic acid, an antibody, or anantigen.

In different embodiments, the dideoxynucleotide comprises a cytosine ora thymine with a 5-position, or an adenine or a guanine with a7-position, and the linker is attached to the 5-position of cytosine orthymine or to the 7-position of adenine or guanine.

The invention provides for the use of any of the linkers describedherein in single nucleotide polymorphism detection using massspectrometry, wherein the linker increases mass separation betweendifferent dideoxynucleotides and increases mass spectrometry resolution.

Labeled dideoxynucleotides are provided which comprise a chemical moietyattached via a linker to a 5-position of cytosine or thymine or to a7-position of adenine or guanine.

In one embodiment, the dideoxynucleotide is selected from the groupconsisting of 2′,3′-dideoxyadenosine 5′-triphosphate (ddATP),2′,3′-dideoxyguanosine 5′-triphosphate (ddGTP), 2′,3′-dideoxycytidine5′-triphosphate (ddCTP), and 2′,3′-dideoxythymidine 5′-triphosphate(ddTTP).

In different embodiments, the linker can comprise a chain structure, ora structure comprising one or more rings, or a structure comprising achain and one or more rings. In different embodiments, the linker iscleavable by a means selected from the group consisting of one or moreof a physical means, a chemical means, a physical chemical means, heat,and light. In one embodiment, the linker is cleavable by ultravioletlight. In different embodiments, the linker is cleavable by ammoniumhydroxide, formamide, or a change in pH -log [H⁺ concentration].

In different embodiments of the labeled dideoxynucleotide, the chemicalmoiety comprises biotin, streptavidin, phenylboronic acid,salicylhydroxamic acid, an antibody, or an antigen.

In one embodiment, the labeled dideoxynucleotide is selected from thegroup consisting of:

-   -   wherein ddNTP1, ddNTP2, ddNTP3, and ddNTP4 represent four        different dideoxynucleotides, or their analogues.

In one embodiment, the labeled dideoxynucleotide is selected from thegroup consisting of:

In one embodiment, the labeled dideoxynucleotide is selected from thegroup consisting of:

wherein ddNTP1, ddNTP2T ddNTP3, and ddNTP4 represent four differentdideoxynucleotides, or their analogues.

In one embodiment, the labeled dideoxynucleotide is selected from thegroup consisting of:

In one embodiment, the labeled dideoxynucleotide has a molecular weightof 844, 977, 1,017, or 1,051. In one embodiment, the labeleddideoxynucleotide has a molecular weight of 1,049, 1,182, 1,222, or1,257. Other molecular weights with sufficient mass differences to allowresolution in mass spectrometry can also be used.

In one embodiment the mass spectrometry is matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

A system is provided for separating a chemical moiety from othercomponents in a sample in solution, which comprises:

-   -   (a) a channel coated with a compound that specifically interacts        with the chemical moiety at the 3′ end of the DNA fragment,        wherein the channel comprises a plurality of ends;    -   (b) a plurality of wells each suitable for holding the sample;    -   (c) a connection between each end of the channel and a well; and    -   (d) a means for moving the sample through the channel between        wells.

In one embodiment of the system, the interaction between the chemicalmoiety and the compound coating the surface is a biotin-streptavidininteraction, a phenylboronic acid-salicylhydroxamic acid interaction, oran antigen-antibody interaction.

In one embodiment, the chemical moiety is a biotinylated moiety and thechannel is a streptavidin-coated silica glass channel. In oneembodiment, the biotinylated moiety is a biotinylated DNA fragment.

In one embodiment, the chemical moiety can be freed from the surface bydisrupting the interaction between the chemical moiety and the compoundcoating the surface. In different embodiments, the interaction can bedisrupted by a means selected from the group consisting of one or moreof a physical means, a chemical means, a physical chemical means, heat,and light. In different embodiments, the interaction can be disrupted byammonium hydroxide, formamide, or a change in pH -log [H⁺concentration].

In one embodiment, the chemical moiety is attached via a linker toanother chemical compound. In one embodiment, the other chemicalcompound is a DNA fragment. In one embodiment, the linker is cleavableby a means selected from the group consisting of one or more of aphysical means, a chemical means, a physical chemical means, heat, andlight. In one embodiment, the channel is transparent to ultravioletlight and the linker is cleavable by ultraviolet light. Cleaving thelinker frees the DNA fragment or other chemical compound from thechemical moiety which remains captured on the surface.

Multi-channel systems are provided which comprise a plurality of any ofthe single channel systems disclosed herein. In one embodiment, thechannels are in a chip. In one embodiment, the multi-channel systemcomprises 96 channels in a chip. Chips can also be used with fewer orgreater than 96 channels.

The invention provides for the use of any of the separation systemsdescribed herein for single nucleotide polymorphism detection.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

Experimental Set I

A. Materials and Methods

PCR amplification. DNA templates containing the polymorphic sites forthe human hereditary hemochromatosis gene HFE were amplified fromgenomic DNA in a total volume of 10 μl, that contains 20 ng of genomicDNA, 500 pmol each of forward (C282Y; 5′-CTACCCCCAGAACATCACC-3′ (SEQ IDNO: 1), H63D; 5′-GCACTACCTCTTCATGGGTGCC-3′ (SEQ ID NO: 2)) and reverse(C282Y; 5′-CATCAGTCACATACCCCA-3′ (SEQ ID NO: 3), H63D;5′-CAGTGAACATGTGATCCCACCC-3′ (SEQ ID NO: 4)) primers, 25 μM dNTPs(Amersham Biosciences, Piscataway, N.J.), 1 U Tag polymerase (LifeTechnologies, Rockville, Md.), and 1× PCR buffer (50 mM KCl, 1.5 mMMgCl₂, 10 mM Tris-HCl). PCR amplification reactions were started at 94°C. for 4 min, followed by 45 cycles of 94° C. for 30 s, 59° C. for 30 sand 72° C. for 10 s, and finished with an additional extension step of72° C. for 6 min. Excess primers and dNTPs were degraded by adding 2 Ushrimp alkaline phosphatase (Roche Diagnostics, Indianapolis, Ind.) andE. Coli exonuclease I (Boehringer Mannheim, Indianapolis, Ind.) in 1×shrimp alkaline phosphatase buffer. The reaction mixture was incubatedat 37° C. for 45 min followed by enzyme inactivation at 95° C. for 15min.

Single base extension using biotin-ddNTPs. The synthetic DNA templatescontaining six nucleotide variations in p53 gene and the five primersfor detecting these variations are shown in Table 1. Theseoligonucleotides and an internal mass standard (5′-TTTTTCTTTTTCT-3′ (SEQID NO: 5), MW=3855 Da) for MALDI-TOF MS measurement were made using anExpedite nucleic acid synthesizer (Applied Biosystems, Foster City,Calif.). SBE reactions contained 20 pmol of primer, 10 pmol ofbiotin-11-ddATP, 20 pmol of biotin-11-ddGTP, 40 pmol of biotin-11-ddCTP(New England Nuclear Life Science, Boston, Mass.), 80 pmol ofbiotin-16-ddUTP (Enzo Diagnostics, Inc., Farmingdale, N.Y.), 2 μl ThermoSequenase reaction buffer, 1 U Thermo Sequenase in its diluted buffer(Amersham Biosciences) and 20 pmol of either synthetic template or 10 μlPCR product in a total reaction volume of 20 μl. For SBE using synthetictemplate 1, 10 pmol of both wild type and mutated templates werecombined with 20 pmol of primers 1 and 3 or 20 pmol of primers 2 and 4.The SBE reaction of primer 5 was performed with template 2 in a separatetube. PCR products from the HFE gene were mixed with 20 pmol of thecorresponding primers 5′-GGGGAAGAGCAGAGATATACGT-3′ (SEQ ID NO: 6)(C282Y) and 5′-GGGGCTCCACACGGCGACTCTC-AT-3′ (SEQ ID NO: 7) (H63D) in SBEto detect the two heterozygous genotypes. All extension reactions werethermalcycled for 35 cycles at 94° C. for 10 s and 49° C. for 30 s.

Solid phase purification. 20 μl of the streptavidin-coated magneticbeads (Seradyn, Ramsey, Minn.) were washed with modified binding andwashing (B/W) buffer (0.5 mM Tris-HCl buffer, 2 M NH₄Cl, 1 mM EDTA, pH7.0) and resuspended in 20 μl modified B/W buffer. Extension reactionmixtures of primers 1-4 with template 1 and primer 5 with template 2were mixed in a 2:1 ratio, while extension reaction mixtures from thePCR products of HFE gene were mixed in equal amounts. 20 μl of eachmixed extension product was added to the suspended beads and incubatedfor 1 hour. After capture, the beads were washed twice with modified B/Wbuffer, twice with 0.2 M triethyl ammonium acetate (TEAA) buffer andtwice with deionized water. The primer extension products were releasedfrom the magnetic beads by treatment with 8 μl 98% formamide solutioncontaining 2% 0.2 M TEAA buffer at 94° C. for 5 min. The released primerextension products were precipitated with 100% ethanol at 4° C. for 30min, and centrifuged at 4° C. and 14000 RPM for 35 min.

MALDI-TOF MS analysis. The purified primer extension is products weredried and re-suspended in 1 μl deionized water and 2 μl matrix solution.The matrix solution was made by dissolving 35 mg of 3-hydroxypicolinicacid (3-HPA; Aldrich, Milwaukee, Wis.) and 6 mg of ammonium citrate(Aldrich) in 0.8 ml of 50% acetonitrile. 10 pmol internal mass standardin 1 μl of 50% acetonitrile was then added to the sample. 0.5 μl of thismixture containing the primer extension products and internal standardwas spotted on a stainless steel sample plate, air-dried and analyzedusing an Applied Biosystems Voyager DE Pro MALDI-TOF mass spectrometer.All measurements were taken in linear positive ion mode with a 25 kVaccelerating voltage, a 94% grid voltage and a 350 ns delay time. Theobtained spectra were processed using the Voyager data analysis package.

B. Detection of Single Nucleotide Polymorphism Using BiotinylatedDideoxynucleotides and Mass Spectrometry

Solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs)were used in single base extension for multiplex genotyping by massspectrometry (MS). In this method, oligonucleotide primers that havedifferent molecular weights and that are specific to the polymorphicsites in the DNA template are extended with biotin-ddNTPs by DNApolymerase to generate 3′-biotinylated DNA extension products (FIG. 1).These products are then captured by streptavidin-coated solid phasemagnetic beads, while the unextended primers and other components in thereaction are washed away. The pure extension DNA products aresubsequently released from the solid phase and analyzed withmatrix-assisted laser desorption/ionization time-of-flight MS. The massof the extension DNA products is determined using a stableoligonucleotide as a common internal mass standard. Since only the pureextension DNA products are introduced to MS for analysis, the resultingmass spectrum is free of non-extended primer peaks and their associateddimers, which increases the accuracy and scope of multiplexing in singlenucleotide polymorphism (SNP) analysis. The solid phase purificationapproach also facilitates desalting of the captured oligonucleotides,which is essential for accurate mass measurement by MS.

Four biotin-ddNTPs with distinct molecular weights were selected togenerate extension products that have a two-fold increase in massdifference compared to that with conventional ddNTPs. This increase inmass difference provides improved resolution and accuracy in detectingheterozygotes in the mass spectrum.

The “lock and key” functionality of biotin and streptavidin is oftenutilized in biological sample preparation as a way to remove undesiredimpurities (23). In different embodiments of the methods describedherein, affinity systems other than biotin-streptavidin can be used.Such affinity systems include but are not limited to phenylboronicacid-salicylhydroxamic acid (31) and antigen-antibody systems.

The multiplex genotyping approach was validated by detecting sixnucleotide variations from synthetic DNA templates that mimic mutationsin exons 7 and 8 of the p53 gene. Sequences of the templates and thecorresponding primers are shown in Table 1 along with the masses of theprimers and their extension products. The mass increase of the resultingsingle base extension products in comparison with the primers is 665 Dafor addition of biotin-ddCTP, 688 Da for addition of biotin-ddATP, 704Da for addition of biotin-ddGTP and 754 Da for addition of biotin-ddUTP.The mass data in Table 1 indicate that the smallest mass differenceamong any possible extensions of a primer is 16 Da (between biotin-ddATPand biotin-ddGTP). This is a substantial increase over the smallest massdifference between extension products using standard ddNTPs (9 Dabetween ddATP and ddTTP). This mass increase yields improved resolutionof the peaks in the mass spectrum. Increased mass difference in ddNTPsfosters accurate detection of heterozygous genotypes (15), since an A/Theterozygote with a mass difference of 9 Da using conventional ddNTPscan not be well resolved in the MALDI-TOF mass spectra. The five primersfor each polymorphic site were designed to produce extension productswithout overlapping masses. Primers extended by biotin-ddNTPs werepurified and analyzed by MALDI-TOF MS according to the scheme in FIG. 1.Extension products of all five primers were well-resolved in the massspectrum free from any unextended primers (FIG. 2A), allowing eachnucleotide variation to be unambiguously identified. Unextended primersoccupy the mass range in the mass spectrum decreasing the scope ofmultiplexing, and excess primers can dimerize to form false peaks in themass spectrum (21). The excess primers and their associated dimers alsocompete for the ion current, reducing the detection sensitivity of MSfor the desired DNA fragments. These complications were completelyremoved by carrying out SBE using biotin-ddNTPs and solid phase capture.Extension products for all four biotin-ddNTPs were clearly detected withwell resolved mass values. The relative masses of the primer extensionproducts in comparison to the internal mass standard revealed theidentity of each nucleotide at the polymorphic site. In the case ofheterozygous genotypes, two peaks, one corresponding to each allele(C/A), are clearly distinguishable in the mass spectrum shown in FIG.2A. TABLE 1 Oligonucleotide primers and synthetic DNA templates fordetecting mutations in the p53 gene. Masses of single base extensionproducts (Da) Biotin- Biotin- Biotin- Biotin- Masses ddcTP ddATP ddGTPddUTP Primers Primer sequences (Da) Δ665 Δ688 Δ704 Δ754 15′-AGAGGATCCAACCGAGAC-3′ 1656 2321 2344 2360 2410 25′-TGGTGGTAGGTGATGTTGATGTA-3′ 3350 4015 4038 4054 4103 35′-CACATTGTCAAGGACGTACCCG-3′ 2833 3498 3521 3538 3587 45′-TACCCGCCGTACTTGGCCTC-3′ 2134 2799 2822 2838 2480 55′-TCCACGCACAAACACGGACAG-3′ 2507 3172 3195 3211 3261 Templates Templatesequences 15′-TACCCG/TGAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3′ (SEQ ID NO:13) 2GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTTGTGCGTGGAGTTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTTTCTCTTCGTTCTCCGT-3′ (SEQ ID NO:14)(Top) The sequences and the calculated masses of primers and the fourpossible single base extension products relative to the internal massstandard are listed. The bold numbers refer to the nucleotide variationsdetected in the p53 gene. (Bottom) The six nucleotide variations intemplate 1 and 2 are shown in bold letters. Template 1 contains aheterozygous genotype (G/T). Primers 1-5 = SEQ ID NOs: 8-12,respectively.

One advantage of MALDI-TOF MS in comparison to other detectiontechniques is its ability to simultaneously measure masses of DNAfragments over a certain range.

In order to explore this feature to detect multiple SNPs in a singlespectrum, if unextended primers are not removed, masses of all primersand their extension products must have sufficient differences to yieldadequately resolved peaks in the mass spectrum. Ross et al.simultaneously detected multiple SNPs by carefully tuning the masses ofall primers and extension products so that they would lie in the rangeof 4.5 kDa and 7.6 kDa without overlapping (14). Since the unextendedprimers occupy the mass range in the mass spectrum, by eliminating them,the approach disclosed herein will significantly increase the scope ofmultiplexing in SNP analysis.

To demonstrate the ability of this method to discriminate SNPs ingenomic DNA, two disease associated SNPs were genotyped in the humanhereditary hemochromatosis (HHC) gene HFE. HHC is a common geneticcondition in Caucasians with approximately 1/400 Caucasians homozygousfor the C282Y mutation leading to iron overload and potentially liverfailure, diabetes and depression (22). A subset of individuals who arecompound heterozygotes for the C282Y and H63D mutations also manifestiron overload. Because of the high prevalence of these mutations and theability to prevent disease manifestations by phlebotomy, accuratemethods for genotyping these two SNPs will foster genetic screening forthis condition. Two PCR products were generated from human genomic DNAfor the C282Y and H63D polymorphic sites of the HFE gene and then usedthese products for SBE with biotin-ddNTPs. After the extension reaction,products were purified using solid phase capture according to the schemein FIG. 1 and analyzed by MALDI-TOF MS. The mass spectrum obtained fromthis experiment is shown in FIG. 2B. Extension products of each primerwere readily identified by their mass relative to the internal massstandard. Heterozygous genotypes of A/G and C/G with a mass differenceof 16 Da and 39 Da respectively were accurately detected at the C282Yand H63D polymorphic sites.

These results indicate that the use of solid phase capturablebiotin-ddNTPs in SBE, coupled with MALDI-TOF MS detection, provides arapid and accurate method for multiplex SNP detection over broad massranges and should greatly increase the number of SNPs that can bedetected simultaneously. In multiplex SBE reactions, the oligonucleotideprimers and their dideoxynucleotide extension products differ by onlyone base pair, which requires analytical techniques with high resolutionto resolve. In addition, a primer designed to detect one polymorphismand an extension product from another polymorphic site may have the samesize, which can not be separated by electrophoresis and otherconventional chromatographic or size exclusion methods. Methods forpurifying DNA samples using the strong interaction of biotin andstreptavidin are widely used (23-27). By introducing the biotin moietyat the 3′ end of DNA, the solid phase based affinity purificationapproach described here is a unique and effective method to remove theoligonucleotide primers from the dideoxynucleotide extension products.

To increase the stability of DNA fragments for MALDI-TOF MS measurementin multiplex SNP analysis, nucleotide analogues (28) and peptide nucleicacid (9) can be used in the construction of the oligonucleotide primers.It has been shown that MALDI-TOF MS could detect DNA fragments up to 100bp with sufficient resolution (29). The mass difference between eachadjacent DNA fragment is approximately 300 Da. Thus, with a massdifference of 100 Da for each primer in designing a multiplex SNPanalysis project, at least 300 SNPs can be analyzed in a single spot ofthe sample plate by MS. It is a routine method now to place 384 spots ineach sample plate in MS analysis. Thus, each plate can produce over100,000 SNPs, which is roughly the entire SNPs in all the coding regionsof the human genome. This level of multiplexing should be achievable bymass tagging the primers with stable chemical groups in SBE usingbiotin-ddNTPs. For SNP sites of interest, a master database of primersand the resulting masses of all four possible extension products can beconstructed. The experimental data from MALDI-TOF MS can then becompared with this database to precisely identify the library of SNPsautomatically. This method coupled with future improvements in massspectrometer detector sensitivity (30) will provide a platform forhigh-throughput SNP identification unrivaled in speed and accuracy.

C. Design and Synthesis of Biotinylated Dideoxynucleotides with MassTags

The ability to distinguish various bases in DNA using mass spectrometryis dependent on the mass differences of the bases in the spectra. Forthe above work, the smallest difference in mass between any twonucleotides is 16 daltons (see Table 1). Fei et al. (15) have shown thatusing dye-labeled ddNTP paired with a regular dNTP to space out the massdifference, an increase in the detection resolution in a singlenucleotide extension assay can be achieved. To enhance the ability todistinguish peaks in the spectra, the current application disclosessystematic modification of the biotinylated dideoxynucleotides byincorporating mass linkers assembled using 4-aminomethyl benzoic acidderivatives to increase the mass separation of the individual bases. Themass linkers can be modified by incorporating one or two fluorine atomsto further space out the mass differences between the nucleotides. Thestructures of four biotinylated ddNTPs are shown in FIG. 3.ddCTP-11-biotin is commercially available (New England Nuclear, Boston).ddTTP-Linker I-11-Biotin, ddATP-Linker II-11-Biotin and ddGTP-LinkerIII-11-Biotin are synthesized as shown, for example, for ddATP-LinkerII-11-Biotin in FIG. 5. In designing these mass tag linker modifiedbiotinylated ddNTPs, the linkers are attached to the 5-position on thepyrimidine bases (C and T), and to the 7-position on the purines (A andG) for subsequent conjugation with biotin. It has been established thatmodification of these positions on the bases in the nucleotides, evenwith bulky energy transfer fluorescent dyes, still allows efficientincorporation of the modified nucleotides into the DNA strand by DNApolymerase (32, 33). Thus, the ddNTPs-Linker-11-biotin can beincorporated into the growing strand by the polymerase in DNA sequencingreactions. Larger mass separations will greatly aid in longer readlengths where signal intensity is smaller and resolution is lower. Thesmallest mass difference between two individual bases is over threetimes as great in the mass tagged biotinylated ddNTPs compared to normalddNTPs and more than double that achieved by the standard biotinylatedddNTPs as shown in Table 2. TABLE 2 Relative mass differences (daltons)of dideoxynucleotides using ddCTP as a reference. CommercialBiotinylated Standard Biotinylated ddNTP with Base ddNTP ddNTP mass taglinker C relative to C  0  0  0 (no linker) T relative to C 15 89 (16linker) 125 (Linker I) A relative to C 24 24 165 (Linker II) G relativeto C 40 40 200 (Linker III) Smallest relative  9 16  35 difference

Three 4-aminomethyl benzoic acid derivatives Linker I, Linker II andLinker III are designed as mass tags as well as linkers-for bridgingbiotin to the corresponding dideoxynucleotides. The synthesis of LinkerII (FIG. 4) is described here to illustrate the synthetic procedure.3-Fluoro-4-aminomethyl benzoic acid that can be easily prepared viapublished procedures (41, 42) is first protected with trifluoroaceticanhydride, then converted to N-hydroxysuccinimide (NHS) ester withdisuccinimidylcarbonate in the presence of diisopropylethylamine. Theresulting NHS ester is subsequently coupled with commercially availablepropargylamine to form the desired compound, Linker II. Using ananalogous procedure, Linker I and Linker III can be easily constructed.

FIG. 5 describes the scheme required to prepare biotinylatedddATP-Linker II-11-Biotin using well-established procedures (34-36).7-I-ddA is coupled with linker II in the presence oftetrakis(triphenylphosphine) palladium(0) to produce 7-Linker II-ddA,which is phosphorylated with POCl₃ in butylammonium pyrophosphate (37).After removing the trifluoroacetyl group with ammonium hydroxide,7-Linker II-ddATP is produced, which then couples withsulfo-NHS-LC-Biotin (Pierce, Rockford Ill.) to yield the desiredddATP-Linker II-11-Biotin. Similarly, ddTTP-Linker I-11-Biotin, andddGTP-Linker III-11-Biotin can be synthesized.

D. Design and Synthesis of Mass Tagged ddNTPs Containing PhotocleavableBiotin

A schematic of capture and cleavage of the photocleavable linker on thestreptavidin coated porous surface is shown in FIG. 6. At the end of thereaction, the reaction mixture consists of excess primers, enzymes,salts, false stops, and the desired DNA fragment. This reaction mixtureis passed over a streptavidin-coated surface and allowed to incubate.The biotinylated fragments are captured by the streptavidin surface,while everything else in the mixture is washed away. Then the fragmentsare released into solution by cleaving the photocleavable linker withnear ultraviolet (UV) light, while the biotin remains attached to thestreptavidin that is covalently bound to the surface. The pure DNAfragments can then be crystallized in matrix solution and analyzed bymass spectrometry. It is advantageous to cleave the biotin moiety sinceit contains sulfur which has several relatively abundant isotopes. Therest of the DNA fragments and linkers contain only carbon, nitrogen,hydrogen, oxygen, fluorine and phosphorous, whose dominant isotopes arefound with a relative abundance of 99% to 100%. This allows highresolution mass spectra to be obtained. The photocleavage mechanism (38,39) is shown in FIG. 7. Upon irradiation with ultraviolet light at300-350 nm, the light sensitive o-nitroaromatic carbonamidefunctionality on DNA fragment 1 is cleaved, producing DNA fragment 2,PC-biotin and carbon dioxide. The partial chemical linker remaining onDNA fragment 2 is stable for detection by mass spectrometry.

Four new biotinylated ddNTPs disclosed here, ddCTP-PC-Biotin,ddTTP-Linker I-PC-Biotin, ddATP-Linker II-PC-Biotin and ddGTP-LinkerIII-PC-Biotin are shown in FIG. 8. These compounds are synthesized by asimilar chemistry as shown for the synthesis of ddATP-LinkerII-11-Biotin in FIG. 6. The only difference is that in the finalcoupling step NHS-PC-LC-Biotin (Pierce, Rockford Ill.) is used, as shownin FIG. 9. The photocleavable linkers disclosed here allow the use ofsolid phase capturable terminators and mass spectrometry to be turnedinto a high throughput technique for DNA analysis.

E. Overview of Capturing a DNA Fragment Terminated With a ddNTP on aSurface and Freeing the ddNTP and DNA Fragment

The DNA fragment is terminated with a dideoxynucleoside monophosphate(ddNMP). The ddNMP is attached via a linker to a chemical moiety (“X” inFIG. 10). The DNA fragment terminated with ddNMP is captured on thesurface through interaction between chemical moiety “X” and a compoundon or attached to the surface (“Y” in FIG. 10). The present applicationdiscloses two methods for freeing the captured DNA fragment terminatedwith ddNMP. In the situation illustrated in the lower part of FIG. 10,the DNA fragment terminated with ddNMP is freed from the surface bydisrupting or breaking the interaction between chemical moiety “X” andcompound “Y”. In the upper part of FIG. 10, the DNA fragment terminatedwith ddNMP is attached to chemical moiety “X” via a cleavable linkerwhich can be cleaved to free the DNA fragment terminated with ddNMP.

Different moieties and compounds can be used for the “X”-“Y” affinitysystem, which include but are not limited to, biotin-streptavidin,phenylboronic acid-salicylhydroxamic acid (31), and antigen-antibodysystems.

In different embodiments, the cleavable linker can be cleaved and the“X”-“Y” interaction can be disrupted by a means selected from the groupconsisting of one or more of a physical means, a chemical means, aphysical chemical means, heat, and light. In one embodiment, ultravioletlight can be used to cleave the cleavable linker. Chemical meansinclude, but are not limited to, ammonium hydroxide (40), formamide, ora change in pH (-log H⁺ concentration) of the solution.

F. High Density Streptavidin-Coated, Porous Silica Channel System.

Streptavidin coated magnetic beads are not ideal for using thephotocleavable biotin capture and release process for DNA fragments,since they are not transparent to UV light. Therefore, the photocleavagereaction is not efficient. For efficient capture of the biotinylatedfragments, a high-density surface coated with streptavidin is essential.It is known that the commercially available 96-well streptavidin coatedplates cannot provide a sufficient surface area for efficient capture ofthe biotinylated DNA fragments. Disclosed in this application is aporous silica channel system designed to-overcome this limitation.

To increase the surface area available for solid phase capture, porouschannels are coated with a high density of streptavidin. For example,ninety-six (96) porous silica glass channels can be etched into a silicachip (FIG. 11). The surfaces of the channels are modified to containstreptavidin as shown in FIG. 12. The channel is first treated with 0.5M NaOH, washed with water, and then briefly pre-etched with dilutehydrogen fluoride. Upon cleaning with water, the capillary channel iscoated with high density 3-aminopropyltrimethoxysilane in aqueousethanol (43). An excess of disuccinimidyl glutarate inN,N-dimethylformamide (DMF) is then introduced into the capillary toensure a highly efficient conversion of the surface end group to asuccinimidyl ester. Streptavidin is then conjugated with thesuccinimidyl ester to form a high-density surface using excessstreptavidin solution. The resulting 96-channel chip is used as apurification cassette.

A 96-well plate that can be used with biotinylated terminators for DNAanalysis is shown in FIG. 11. In the example shown, each end of achannel is connected to a single well. However, for other applications,the end of a channel could be connected to a plurality of wells.Pressure is applied to drive the samples through a glass capillary intothe channels on the chip. Inside the channels the biotin is captured bythe covalently bound streptavidin. After passing through the channel,the sample enters into a clean plate in the other end of the chip.Pressure applied in reverse drives the sample through the channelmultiple times and ensures a highly efficient solid phase capture. Wateris similarly added to drive out the reaction mixture and thoroughly washthe captured fragments. After washing, the chip is irradiated withultraviolet light to cleave the photosensitive linker and release theDNA fragments. The fragment solution is then driven out of the channeland into a collection plate. After matrix solution is added, the samplesare spotted on a chip and allowed to crystallize for detection byMALDI-TOF mass spectrometry. The purification cassette is cleaned bychemically cleaving the biotin-streptavidin linkage, and is then washedand reused.

Experimental Set II

A. Synopsis

The following experiments show the simultaneous genotyping of 30nucleotide variations in the p53 gene from human tumors in one tube, byusing solid phase capturable dideoxynucleotides to generate single baseextension products which are detected by mass spectrometry. Bothhomozygous and heterozygous genotypes are accurately determined withdigital resolution. This is the highest level of SNP multiplexingreported thus far using mass spectrometry, indicating the approach willhave wide applications in screening a repertoire of genotypes incandidate genes as potential markers for cancer and other diseases.

B. Introduction

With the completion of the Human Genome Project, a stage has been set toscreen genetic mutations for identifying disease genes in a genomewidescale (44). Matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF MS), which allows rapid DNA samplemeasurement yielding digital data, has been explored to detect singlenucleotide polymorphisms (SNPs) using invasive cleavage (11) andprimer-directed base extension (14, 45). Conventional single baseextension (SBE) methods using MS to measure multiplex SNPs requireunambiguous simultaneous detection of a library of primers and theirextension products. However, limitations in resolution and sensitivityof MALDI-TOF MS for longer DNA molecules make it difficult tosimultaneously measure DNA fragments over a large mass range. Therequirement to measure both primers and their extension products in thisrange limits the scope of multiplexing. The use of MALDI-TOF MS andmolecular affinity for multiplex digital SNP detection using solid phasecapturable (SPC) dideoxynucleotides and SBE has recently been explored,establishing the feasibility of simultaneously measuring 20 SNPs insynthetic DNA templates (46). This study shows the simultaneousgenotyping of 30 nucleotide variations, corresponding to known sites ofcancer-associated somatic mutations, in exons 5, 7 and 8 of the p53 genefrom human tumors in one tube using the SPC-SBE method. This is thehighest level of multiplexing reported thus far using mass spectrometryfor SNP analysis.

C. Materials and Methods

Multiplex PCR and single base extension reactions Multiplex PCR wasperformed to amplify 3 regions in exons 5, 7 and 8 of the p53 gene. Theprimers for each region were 5′-TATCTGTTCACTTGTGCCC-3′ (exon 5,forward), 5′-CAGAGGCCTGGGGA-CCCTG-3′(exon 5, reverse),5′-CTGCTTGCCACAGGTCTC-3′(exon 7, forward), 5′-CACAGCAG-GCCAGTGTGC-3′(exon 7, reverse), 5¹-GGACCTGATTTCCTTAC-TG-3′ (exon 8, forward), and5′-TGAATCTGAGGCATAACTG-3′ (exon 8, reverse). The 45 1 PCR reactionconsisted of 180 ng genomic DNA, 1.5 nmol dNTP, 4.5 1 10× PCR buffer, 15mM MgCl₂, 4 pmol of forward and reverse primers for exons 5 and 7, 6pmol of forward and reverse primers for exon 8, and 1.0 U of JumpStartRedAccuTaq DNA Polymerase. After a 5 min 96° C. hot start, the touchdownPCR program was performed with 10 cycles of 96° C. (30 sec), 67° C. to57° C. (−1.0° C. per cycle, 30 sec) and 72° C. (30 sec), an additional30 cycles of 96° C. (30 sec), 57° C. (30 sec) and 72° C. (30 sec), and afinal extension at 72° C. for 7 min. The 30 SBE primers (Table 3) weredesigned to yield extension products with a sufficient mass differenceand to be extended simultaneously in a single tube. Primer sequenceswere designed to avoid any overlap in mass, and the formation ofsecondary structures. To evenly separate the masses of such a largenumber of primers for SBE, some primers were synthesized using methyl-dCand dU phosphoramidites (Glen Research) to replace dC and dTrespectively. Substitution of dC by methyl-dC increased the primer massby 14 Da whereas a change from dT to dU decreased the mass by 14 Da.Primers were synthesized using an Applied Biosystems DNA synthesizer.The procedures for the SBE, solid phase purification and MALDI-TOF MSmeasurement were performed as described (Kim et al., AnalyticalBiochemistry 2003, 316, 251). Direct DNA sequencing was conducted usingenergy transfer terminator chemistry and a MegaBACE 1000 capillary DNAsequencer (Amersham Bioscience).

D. Discussion

Thirty polymorphic sites, including the most frequently mutated p53codons, were chosen to explore the high multiplexing scope of theSPC-SBE method (FIG. 1). Thirty primers specific to each polymorphicsite were designed to yield SBE products with sufficient massdifferences. This was achieved by tuning the mass of some primers usingmethyl-dc and dU to replace dC and dT, respectively. Human genomic DNAwas amplified by multiplex PCR to produce amplicons of three p53 exons.The 30 primers were mixed with the PCR products and biotinylateddideoxynucleotides for SBE to generate 3′-biotinylated extension DNAproducts. These products were then captured by streptavidin-coated solidphase magnetic beads, while the unextended primers and other componentsin the reaction were washed away. The pure DNA products weresubsequently released from the solid phase and analyzed by MALDI-TOF MS.The nucleotide at the polymorphic site is accurately identified by themass of the DNA extension product in a mass spectrum. Since only the DNAextension products are isolated for MS analysis, the resulting massspectrum is free of non-extended primer peaks and their associateddimers, increasing accuracy and scope of multiplexing. The solid phasepurification also facilitates desalting of the captured DNA, a processthat is critical for accurate mass measurement by MALDI-TOF MS.

The SPC-SBE genotyping approach was used to analyze nucleotidevariations in 30 codons of 3 exons of the p53 gene from 30 Wilms'tumors, 19 head and neck squamous carcinomas and 3 colorectalcarcinomas. Primer sequences are shown in Table 3 along with the massesof the primers and their extension products. Extension products of all30 primers were resolved in the mass spectrum, free from any unextendedprimers, yielding digital data to unambiguously determine eachnucleotide variation (FIGS. 13A-13C). Unextended primers occupy the massrange in the mass spectrum decreasing the scope of multiplexing, andexcess primers can dimerize to form false peaks in the mass spectrum(21). The excess primers and their associated dimers also compete forthe ion current, reducing the detection sensitivity of MS for thedesired DNA fragments. These complications were completely removed inthe SPC-SBE method. When using conventional ddNTPs, the mass differencebetween ddATP and ddTTP is 9 Da, which is difficult to resolve byMALDI-TOF MS (15). In the SPC-SBE method using biotinylated ddNTPs, thedifference between A and T is increased to 66 Da, which fosters accuratedetection of heterozygous genotypes.

None of the 30 Wilms' tumor samples showed somatic mutations for the 30polymorphic sites tested, yielding 30 distinct peaks corresponding tothe wild type p53 sequences in a mass spectrum (FIG. 13A). In contrast,two of the 19 head and neck tumor samples contained a genetic variation;one at codon 157 (G/T heterozygous configuration; primary tumor biopsy;FIG. 13B) and the other at codon 151 (C to T homozygous; squamouscarcinoma cell line; FIG. 14). In the three colorectal tumor cell linestested, one (HCT-116) had 30 wild type p53 sequences for the 30 sites,yielding a mass spectrum similar to the one shown in FIG. 13A, while theother two (HT-29 and SW-480) had a G to A homozygous mutation in codon273 (FIG. 13C). Both heterozygous and homozygous genotypes were clearlydetected in the 30 codons with great accuracy. The G/T heterozygote(4684/4734 Da) was shown with two peaks corresponding to the wild typeand mutant alleles, respectively (FIG. 13B). These data, confirmed bydirect DNA sequencing, are consistent with the known paucity of the p53mutations in Wilms' tumor, and the known occurrence of such mutations insquamous carcinomas and colorectal carcinomas.

It has been reported that MALDI-TOF MS could detect DNA sequencingfragments up to 100 bp with sufficient resolution using cleavableprimers (29). The mass difference between each adjacent DNA sequencingfragment is approximately 300 Da. In principle, with a mass differenceof 100 Da for each primer in designing a multiplex SNP analysis projectusing the SPC-SBE method, at least 300 SNPs can be analyzed in a singlespot of an MS sample plate. Thus, each MS sample plate with 384 spotscan produce over 100,000 SNPs, which is roughly the number of tag SNPsrequired to identify all the haplotypes in the human genome. This levelof multiplexing should be achievable by mass tuning the primers withnucleotide analogues containing stable chemical groups (28). It isanticipated that the SPC-SBE high-throughput digital SNP detectionapproach will have wide applications in screening a repertoire ofgenotypes in candidate genes as potential markers for cancer and otherdiseases. TABLE 3 Thirty p53 codons and the corresponding 30 SBEprimers. Mass of Single Base Primer Primer Extention Products (Da)Number Exon Codon Sequences (5′-3′) Modification Mass (Da) ddATP-BddCTP-B ddGTP-B ddUTP-B 1 5 179 (CAT) GCGCTGCCCCCAC None 3857 4545 45224561 4611 2 5 157 (GTC) GCCC GGCACCCGC methyl C 3980 4668 4645 4684 47343 5 179 (CAT) GCGCTGCCCCCACC None 4146 4834 4811 4850 4900 4 5 163 (TAC)CGCCATGGCCATCT methyl C 4270 4958 4935 4974 5024 5 5 158 (CGC)CCGGCACCCGCGTCC None 4475 5163 5140 5179 5229 6 7 248 (CGG)TGGGCGGCATGAACC None 4618 5306 5283 5322 5372 7 5 132 (AAG)TCCCCTGCCCTCAACA methyl C 4736 5424 5401 5440 5490 8 8 298 (GAG)AGGGGAGCCTCACCAC None 4876 5564 5541 5580 5630 9 8 285 (GAG)GAGAGACCGGCGCACA methyl C 4995 5683 5660 5699 5749 10 5 161 (GCC)CCCGCGTCCGCGCCATG None 5108 5796 5773 5812 5862 11 7 249 (AGG)GGCGGCATGAACCGGAG methyl C 5341 6029 6006 6045 6095 12 8 266 (GGA)GTAGTGGTAATCTACTGG dU 5486 6174 6151 6190 6240 13 8 286 (GAA)AGAGACCGGCGCACAGAG methyl C 5638 6326 6303 6342 6392 14 7 258 (GAA)CCTCACCATCATCACACTG methyl C 5765 6453 6430 6469 6519 15 5 176 (TGC)ACGGAGGTTGTGAGGCGCT dU 5897 6585 6562 6601 6651 16 5 152 (CCG)GTGGGTTGATTCCACACCCC dU 6041 6729 6706 6745 6795 17 8 273 (CGT)ACGGAACAGCTTTGAGGTGC None 6182 6870 6847 6886 6936 18 7 234 (TAC)CTGACTGTACCACCATCCACT None 6286 6974 6951 6990 7040 19 7 248 (CGG)TCCTGCATGGGCGGCATGAAC dU 6405 7093 7070 7109 7159 20 7 249 (AGG)GCATGGGCGGCATGAACCGGA None 6521 7209 7186 7225 7275 21 8 282 (CGG)TTGTGCCTGTCCTGGGAGAGAC dU 6698 7386 7363 7402 7452 22 8 278 (CCT)TGAGGTGCGTGTTTGTGCCTGT None 6819 7507 7484 7523 7573 23 5 135 (TGC)CCCTGCCCTCAACAAGATGTTTT None 6935 7623 7600 7639 7689 24 7 245 (GGC)TGTGTAACAGTTCCTGCATGGGC dU 7043 7731 7708 7747 7797 25 7 237 (ATG)TACCACCATCCACTACAACTACAT None 7170 7858 7835 7874 7924 26 7 242 (TGC)ACAAC TACATGTGTAACAGTTCCT dU 7282 7970 7947 7986 8036 27 7 241 (TCC)ACTACAACTACATGTGTAACAGTT methyl C 7390 8078 8055 8094 8144 28 8 275(TGT) GGAACAGCTTTGAGGTGCGTGTTT methyl C 7497 8185 8162 8201 8251 29 5141 (TGC) ATGTTTTGCCAACTGGCCAAGACCT None 7617 8305 8282 8321 8371 30 5175 (CGC) CAGCACATGACGGAGGTTGTGAGGC None 7772 8460 8437 8476 8526The position of the nucleotide variation tested in each codon is shownin bold. The primer sequence and modification is specified and themodified nucleotides are shown in bold. The mass of each primer isindicated along with the mass of all four possible SBE products. Themass values in bold specify the wild type nucleotide sequences (ddNTP-B= Biotinylated dideoxynucleotides).

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1. A method for determining the identity of a nucleotide present at apredetermined site in a DNA whose sequence immediately 3′ of suchpredetermined site is known which comprises: (a) treating the DNA withan oligonucleotide primer whose sequence is complementary to such knownsequence so that the oligonucleotide primer hybridizes to the DNA andforms a complex in which the 3′ end of the oligonucleotide primer islocated immediately adjacent to the predetermined site in the DNA; (b)simultaneously contacting the complex from step (a) with four differentlabeled dideoxynucleotides, in the presence of a polymerase underconditions permitting a labeled dideoxynucleotide to be added to the 3′end of the primer so as to generate a labeled single base extendedprimer, wherein each of the four different labeled dideoxynucleotides(i) is complementary to one of the four nucleotides present in the DNAand (ii) has a molecular weight which can be distinguished from themolecular weight of the other three labeled dideoxynucleotides usingmass spectrometry; and (c) determining the difference in molecularweight between the labeled single base extended primer and theoligonucleotide primer so as to identify the dideoxynucleotideincorporated into the single base extended primer and thereby determinethe identity of the nucleotide present at the predetermined site in theDNA.
 2. The method of claim 1, wherein each of the four labeleddideoxynucleotides comprises a chemical moiety attached to thedideoxynucleotide by a different linker which has a molecular weightdifferent from that of each other linker.
 3. The method of claim 1 whichfurther comprises after step (b) the steps of: (i) contacting thelabeled single base extended primer with a surface coated with acompound that specifically interacts with a chemical moiety attached tothe dideoxynucleotide by a linker so as to thereby capture the extendedprimer on the surface; and (ii) treating the labeled single baseextended primer so as to release it from the surface.
 4. The method ofclaim 3 which further comprises after step (i) the step of treating thesurface to remove primers that have not been extended by a labeleddideoxynucleotide.
 5. The method of claim 1, wherein step (c) comprisesdetermining the difference in mass between the labeled single baseextended primer and an internal mass calibration standard added to theextended primer.
 6. The method of claim 3, wherein the interactionbetween the chemical moiety attached to the dideoxynucleotide by thelinker and the compound on the surface comprises a biotin-streptavidininteraction, a phenylboronic acid-salicylhydroxamic acid interaction, oran antigen-antibody interaction.
 7. The method of claim 3, wherein thestep of releasing the labeled single base extended primer from thesurface comprises disrupting the interaction between the chemical moietyattached to the dideoxynucleotide by the linker and the compound on thesurface.
 8. The method of claim 7, wherein the interaction is disruptedby a means selected from the group consisting of one or more of aphysical means, a chemical means, a physical chemical means, heat, andlight.
 9. The method of claim 2, wherein the linker is attached to thedideoxynucleotide at the 5-position of cytosine or thymine or at the7-position of adenine or guanine.
 10. The method of claim 3, wherein thestep of releasing the labeled single base extended primer from thesurface comprises cleaving the linker between the chemical moiety andthe dideoxynucleotide.
 11. The method of claim 10, where the linker iscleaved by a means selected from the group consisting of one or more ofa physical means, a chemical means, a physical chemical means, heat, andlight.
 12. The method of claim 11, wherein the linker is cleaved bylight.
 13. The method of claim 2, wherein the linker comprises aderivative of 4-aminomethyl benzoic acid, a 2-nitrobenzyl group, or aderivative of a 2-nitrobenzyl group.
 14. The method of claim 13, whereinthe linker comprises one or more fluorine atoms.
 15. The method of claim14, wherein the linker is selected from the group consisting of:


16. The method of claim 3, wherein the chemical moiety comprises biotin,the labeled dideoxynucleotide is a biotinylated dideoxynucleotide, thelabeled single base extended primer is a biotinylated single baseextended primer, and the surface is a streptavidin-coated solid surface.17. The method of claim 16, wherein the biotinylated dideoxynucleotideis selected from the aroup consisting of ddATP-11-biotin,ddCTP-11-biotin, ddGTP-11-biotin, and ddTTP-16-biotin.
 18. The method ofclaim 16, wherein the biotinylated dideoxynucleotide is selected fromthe group consisting of:

wherein ddNTP1, ddNTP2, ddNTP3, and ddNTP4 represent four differentdideoxynucleotides.
 19. The method of claim 18, wherein the biotinylateddideoxynucleotide is selected from the group consisting of:


20. The method of claim 16, wherein the biotinylated dideoxynucleotideis selected from the group consisting of:

wherein ddNTP1, ddNTP2, ddNTF3, and ddNTF4 represent four differentdideoxynucleotides.
 21. The method of claim 20, wherein the biotinylateddideoxynucleotide is selected from the group consisting of:


22. The method of claim 16, wherein the streptavidin-coated solidsurface is a streptavidin-coated magnetic bead or a streptavidin-coatedsilica glass.
 23. The method of claim 1, wherein steps (a) and (b) areperformed in a single container or in a plurality of connectedcontainers.
 24. A method for determining the identity of nucleotidespresent at a plurality of predetermined sites, which comprises carryingout the method of claim 3 using a plurality of different primers eachhaving a molecular weight different from that of each other primer,wherein a different primer hybridizes adjacent to a differentpredetermined site.
 25. The method of claim 24, wherein differentlinkers each having a molecular weight different from that of each otherlinker are attached to the different dideoxynucleotides to increase massseparation between different labeled single base extended primers andthereby increase mass spectrometry resolution.